DNA encoding variant CD44 surface proteins associated with metastatic tumors

ABSTRACT

The presence of certain extracellular regions (&#34;ECR&#34;) from human and rat variants of the CD44 membrane glycoprotein have been found to be associated with metastasis ability in tumor cells. Isolated polynucleotides encoding the ECRs permit expression of the ECR polypeptide, which in turn can be used as an antigen to obtain monoclonal antibodies that recognize the ECR polypeptide. The anti-ECR monoclonal antibodies have the ability to prevent metastasis by tumor cells that would otherwise metastasize and spread.

The invention concerns variant CD44 surface proteins, antibodies against the variant determinants of these proteins, as well as processes for their production, furthermore the DNA sequences which code for these variant protein fragments, as well as the use of these proteins or parts thereof and the antibodies directed against them for the diagnosis and therapy of tumour metestases.

BACKGROUND OF THE INVENTION

The ability to metastase forms the actual life-endangering property of malignant tumour cells. The original primary tumour cells probably acquire this property by a whole series of changes in the course of the tumour progression. As a result of this process, cancer cell variants are continuously detached from the primary tumour mass, penetrate the extracellular matrix and migrate into the lymphatic system or the blood circulation. Often adhering to one another, the metastasing tumour cells are transported in the blood or lymph system, leave the vascular system at other places in order there to penetrate into secondary tissue and form daughter tumours (survey of Hart et al., 1989; Nicolson, 1987). The formation of metastases requires a whole series of interactions of the tumour cells with intercellular matrix and other cells. Almost all of these interactions require cell surface components, such as e.g. the receptors for matrix and lamina, surfacebound proteolytic enzymes, as well as cell adhesion molecules with inclusion of those which cause organspecific adhesion and thus organ preference of the metastasis, furthermore growth factors and growth factor receptors.

It is known that the membrane proteins differentiate non-metastasing and metastasing tumour cells of the BSp73 rat tumours, demonstrated by antibody reaction (Matzku et al., 1983 and 1989).

SUMMARY OF THE INVENTION

It has now been found that the metastasing BSp73ASML tumour cells contain a surface protein which, in part, corresponds to a known glycoprotein participating in the lymphocyte adhesion and cell-cell and cell-matrix exchange action (designation of the normal glycoprotein in humans: CD44, hermes-1, in the mouse: Ppg-1 and in the rat: HEBFln). However, the new variant CD44 surface protein differs from these known sequences by an extracellular region (ECR) of 154 amino acids which is introduced between the 220th and 237th amino acid of the human CD44 sequence (or 224th and 239th amino acid of the mouse sequence). This new glycoprotein appears to possess an important role for the cell/matrix or cell/cell binding in the case of the metastasis. Therefore, the production and characterisation of this protein region (ECR) forms one of the tasks of the present invention. By immunisation of mice with membrane proteins which have been obtained from BSp73ASML, spleen cells were produced which form antibodies against the ECR of the variant CD44 surface protein. According to the method of Ko hler (1981), these are fused by polyethylene glycol with myeloma cells in order to produce permanent cultures. By means of cloning and selection of those cultures which produce antibodies which react with BSp73ASML but not with the non-metastasing parent form and also not with other non-tumorigenic rat cells, there can be obtained specific antibodies against the new protein part ECR. For the further investigation, a monoclonal antibody was chosen which stains the BSp73ASML cells in the immunofluorescence test especially intensively, which has received the designation mAb1.1ASML (mAb: monoclonal antibody).

In the Western blot test, in a protein hydrolysate frown BSp73ASML, there can be determined 4 protein bands with molecular weights of 120,000; 150,000; 180,000 and 200,000 with mAb1.1.ASML, whereas extracts from rat fibroblast cells and non-metastasing rat tumour cells give no significant reaction. It has not yet been possible to determine whether these size differences are due to a different original amino acid sequence or to a differently strong subsequent protein modification. In any case, the epitope recognised by the antibody is contained in all 4 protein species but not in the proteins serving for the control from the non-metastasing BSp73As cells or from normal rat cells.

Isolation of cDNA sequences which code for the ECR of the surface protein

The monoclonal antibody mAb1.1ASML was used in order to discover the ECR-coding cDNA sequences in a bacterial expression bank. The bank was constructed with the help of PolyA+ RNA from BSp73ASML and the pEX vector system (Stanley & Luzio, 1984). The products coded by the cDNA sequences are found as β-galactosidase fusion proteins with the help of the antibodies. A so-isolated cDNA clone positive for the monoclonal antibody 1.1ASML with the name pEX34 carries a cDNA sequence piece of 167 nucleotides. This cDNA piece was now used in order to pattern through a larger cDNA bank in the vector pSP65, again from BSp73ASML RNA. One of these clones isolated therewith, pM66, served thereto to isolate the total length cDNA clone pMeta-1 with the help of so-called "primer" elongation (starter oligonucleotides) and of the polymerase chain reaction (PCR). Evidence of the total length was obtained with the help of the primary elongation (3207 nucleotides). The colinearity with an RNA from the BSp73ASML tumour was documented by RNase and S1 protection analysis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the structure of a cDNA of the rat surface protein p-Meta-1. Bar "A" represents the extracellular region ("ECR") at positions 941-1108. Bar "B" represents the sequence between positions 1403-1572. Bar "C" represents the sequence between the starting position to position 794. Bar "D" represents sequences at the end of the cDNA clone.

FIGS. 2a-2d depict the sequence of the CD44 protein and DNA from p-Meta-1. The signal peptide is represented by ".sup...... ". The ECR is represented by "-". The transmembrane region is represented by " ".

FIGS. 3a(1)-3a(3) depict the nucleotide and protein sequence of a human extracellular region and compares the protein sequence to a rat sequence, and

FIGS. 3b(1)-3b(2) compare protein sequences of rat rMeta-1, human hCD44 and mouse mCD44.

FIGS. 4A-4G depict patterns of RNA derived from CD44.

FIG. 5 depicts RNA patterns from G418-resistant and p-Meta-1 expressing BSp73AS cells.

The isolation of the cDNAs with the help of the expression in bacteria and the recognition by the antibodies proved that the antibody recognised primary amino acid sequence in the ECR of the surface protein.

ECR-coding messenger RNA is expressed in BSp73ASML but not in BSp73AS cells.

Three different sequence samples were produced from the cDNA clones in order to demonstrate specific messenger RNAs by hybridisation. A sample A covers the cDNA region which codes for the ECR (positions: 941-1108). The sample B represents sequences between the positions 1403-157.2 and sample C carries sequences. from the start up to position 794 from pMeta-L (FIG. 1). Poly A+ RNA of the BSp73 tumour cell line was separated electrophoretically and analysed with the help of RNA transfer hybridisation. Four of the cell lines, which do not metastase, contain no RNA which is homologous to the sample A, whereas RNAs from the metastasing tumour cells strongly react BSp73ASML. In this RNA preparation, the sample A recognises a heterogenous mixture of various RNA sizes between 2.2 and 3.3 kb and a larger RNA species of 4.9 kb. The exclusive expression of the specific membrane proteins recognised by the monoclonal antibody 1.1ASML in the metastasing tumour cell variant is obviously based upon the exclusive expression of the corresponding ECR-coding messenger RNAs. Obviously the complete cDNA clone pMeta-1 with 3.2 kb cannot represent all sequences of this RNA species. It can only represent one species from the heterogenous mixture of RNA sizes. The samples B and C give the same hybridisation pattern as sample A in the separation of the BSp73ASML RNA in any case as far as one can ascertain in the heterogeneity, i.e. these RNA species carry sequences which are complementary to all three samples, A, B and C. In contradistinction to sample A, sample B and C also recognise messenger RNA species in the non-metastasing cell lines. However, the RNA sizes differ clearly from those in BSp73ASML, there are, namely, detected four clearly differentiatable messenger RNA species with the sizes 1.5, 2.0, 2.9 and 4.3 kb. Although these RNA species could be hidden in the heterogenous mixture of the RNAs from BSp73ASML, it is, nevertheless, certain that they do not exist in the same amount in the BSp73ASML. It is decisive that RNA sequences with complementarity to sample A are obviously completely absent in the non-metastasing cells. Therefore, we can carefully conclude that the sequences of the samples A, B and C are contained in the same RNAs in the metastasing tumour, namely, in a manner as though sample A sequences have been spliced into the B-C-positive RNAs and as though this alternative splicing process only occurred in the metastasing cell line.

In order to demonstrate the colinearity between RNA and cDNA and in order to analyse the difference of the RNAs between the BSp73AS and the BSp73ASML cells, S1 nuclease and RNase protection analyses were carried out. The protected DNA or RNA fragments could only be smaller than the total length because they contain 5'-end vector sequences which cannot hybridise with the RNAs from the tumour cells. We consider first the transfer to the 3' side: the transfer of sequences with homology to sample A to those to sample B. Both techniques show a single RNA species in BSp73AS which is colinear with the samples over a wide range. Furthermore, 5' thereof differentiate cDNA or RNA sample, which certainly correspond to RNA sequences from BSp73ASML, from the RNA from the BSp73AS cells. In particular, the RNAs from BSp73ASML contain sequences which protect larger fragments of the samples. The largest fragments correspond to the full length of the DNA or RNA pieces which were offered for the protection analysis. Smaller fragments are also detectable. Since the RNA transfer hybridisations have certainly uncovered a heterogenous mixture of different sizes of RNAs, it is possible that these indicate smaller protected fragments of RNA species which diverge elsewhere from the cDNA, i.e. at positions between the previously detected divergence point and the 5' end of the offered samples. The RNA species are also not detectable in the BSp73AS cells.

We now consider the point of the divergence on the 5' side, thus the transition of sequences which hybridise with the sample C to those which hybridise with the sample A, thus the ECR-coding sequence. The analysis gives corresponding results for the 5' breaking point. RNAs from BSp73AS can protect the offered samples only over a small range. Messenger RNAs from BSp73ASML protect longer fragments. They are, namely, colinear over the whole length of the offered sample. One can thus conclude that the cDNA clone represents pMeta-1 sequences which are distinctive in the metastasing tumour cells BSp73ASML. The 3' and 5' regions are also found in RNAs from BSp73AS. The ECR-coding sequences with the definite transitions, which can be mapped with the help of these above-described techniques, indicate that here an alternative splicing mechanism must be present for the RNA formation. The 5' and 3' breakage points of the transitions to the ECR sequences are marked by arrows in FIG. 1.

The monoclonal antibody 1.1ASML identifies a variant form of the glycoprotein CD44.

In order to obtain structural information about the surface protein, all cloned cDNA molecules have been sequenced. The nucleotide sequence of the total length clone pMeta-1 and the amino acid sequences derived therefrom are shown in FIGS. 2a-2d (SEQ ID NOS 1 and 2). The total length cDNA clone traverses 3207 nucleotides (SEQ ID NO:1). The 3' terminus carries a PolyA end, two additional polyadenylation signals lie at positions 2288 and 1743. The first ATG codon follows a consensus initiation sequence and opens a readiag frame of 1509 nucleotides, corresponding to 503 amino acids (SEQ ID NO:2). As one should assume for a membranestanding protein, the first 21 amino acids are hydrophobic and represent a signal peptide. No part of these sequences is hitherto to be found in the data bases. However, we found sequence homology to the recently published data about the lymphocyte homing receptor CD44 (of Pgp-1) (Idzerda et el., 1989; Goldstein et al., 1989; Stamenkovic et al., 1989; Nottenburg et al., 1989; Zhou et al., 1989). The homologies are strictly limited to the 5' and 3' parts of the cDNA with inclusion of non-translated regions and they end at the already above-mentioned points of divergence between the BSp73AS and the BSp73ASML RNA sequences. The total extent between the divergence points (in FIG. 2 characterised by colour markings), thus the whole extent of the metastasis-specific ECR-coding sequence, is not represented in the Pgp1 or CD44 sequences. The metastasis-specific glycoprotein obviously represents a variant of the CD44 glycoproteins. It carries, namely, an additional extracellular domain of 156 amino acids and thus an expanded extracellular region of 410 amino acids (less 21 amino acid signal peptide), in comparison with 270 amino acids (also less signal peptide) of the unchanged CD44 glycoprotein. However, in the non-metastasing BSp73AS cells, the unchanged forms of this CD44 family are detected. cDNA sequences of these BSp73AS RNAs have also been cloned and the identity with the metastasis-specific clones outside Of the extra domain is demonstrated.

The expression of the variant CD44 is correlated with the metastatic potential.

In order to test whether the expression of the variant CD44 glycoproteins takes place without exception in the BSp73ASML cells and whether it stands in connection with the metastatic potential of these cells or with the metastatic potential in general, we studied a series of isogenic rat tumour cell lines, namely, the tumour cell lines of the mammary carcinoma system 13762 NF (Neri et al., 1982). We here compare cell lines which have been derived from the parental tumour, namely, the MTPa, MTC, MTF7 and MTA cells (group 1), with cell lines which were established from lymph nodes or lung metastases, namely, MTLy, MTLn2, MTLn3 (group 2). The group 1 cells essentially express the normal CD44 pattern similarly to the RNAs from the BSp73AS cells when one hybridises with sample B. On the other hand, with sample A, there is detected a smaller amount of a diffuse RNA band which has about the size 2.5 kb. On the other hand, the group 2 cells show a completely different RNA pattern. Both samples A and B hybridise with larger RNA species. The sizes resemble those which are detected with BSp 73ASML. The similarity is also documented by RNAse and S1 protection analyses. On the basis of these data, we conclude that a change of the splicing pattern of the RNA and the expression of variant CD44 is correlated with the formation of metastases and that the acquired pattern in these metastasing mammary carcinoma cells corresponds very much to those which we have already got to know for the metastasing BSp73ASML cell line. The high molecular proteins recognised by the antibodies correspond to the two high molecular species of proteins which were detected in the BSp73ASML extracts. In this mammary tumour series, we thus discovered also a metastasis-specific expression of RNA species and of high molecular proteins. That in the group 1, thus the so-called parenteral cell lines, any RNAs were found at all which hybridised with the sample A, thus the ECR-coding sequence, and that we can also see a weak coloration of a protein of 100,000 Dalton with the antibody, we attribute to the fact that the group 1 cells also possess small metastasing ability quite contrary to our original cell line BSp73AS which shows no metastasing behaviour at all.

The monoclonal antibody 1.1ASML inhibits matastasis formation in the rat.

In a series of experiments for the metastasis formation of the tumour cell line BSp73ASML in isogenic rats, cells were injected subcutaneously and at different times the monoclonal antibody 1.1ASML was injected intraperitoneally at intervals of two to three days before and after the tumour administration. In the scope of this immunological protocol, it was also determined how the immune response of the rat towards the injected antibody had taken place. There result, namely, anti-mouse immunoglobulin antibodies, as well as also anti-idiotype antibodies. The result of this series of experiments is that the growth and the metastasing of the tumour is considerably delayed by injection of 1.1ASML. This delay permits, in its kinetic, the conclusion that the antibody interferes with a primary process of the metastasing. The experiment shows to us that the protein structure on the surface of the metastasing cells recognised by the antibody has a role in the metastasing process and that therapeutic and diagnostic plans are realistic.

Isolation of the homologous human sequence for the ECR-coding sequence part of the rat cDNA.

For human tumour cells in culture, the possibility naturally does not exist as a matter of course of detecting, correspondingly the rat system, whether they also still retain metastasing properties. Experiments with immune-deficient mice make possible only very limited predictions regarding the metastasis potential in the case of humans. Therefore, relatively many tumour cell lines, which have been taken up in culture anywhere in the world at points of time lying a long time ago, would have to be tested for whether they express the sequences which we could detect for the rat metastases. It has been possible to find such a tumour cell line. It originates from a large-cell lung carcinoma of humans and bears the number: LCLC97. In this tumour cell line can be detected three definite. RNA species (sizes: 5.5; 3.4 and 2.8 kb) which behave quite corresponding to the RNAs which are detectable in the metastasing tumour cell lines of the rat. They hybridise, namely, not only with the sample A but also with the samples B and C, i.e. that also these human RNA species are identical over wide ranges to the cDNA pMeta-1 (85%).

However, the monoclonal antibody 1.1ASML does not react with this tumour cell, i.e. the piece of protein recognised by the antibody must, in the region of the antigen determinant, differ from the proteins which exist on the surface of the human tumour cells. For the non-reactivity, there suffice already the smallest variations on the basis of the high specificity of the antibody. The human tumour cell LCLC97 now served the purpose of constructing a cDNA bank. On the basis of the high agreement between the rat and human sequences, a cDNA clone could be isolated which showed homology with the sample A. The human cDNA was sequenced. In FIGS. 3a(1)-3b(2) is shown the primary sequence and the amino acid sequence derived (SEQ ID NOS 3 and 4) therefrom. One can see that, over large regions, identicity exists between the rat (SEQ ID NO:5) and the human sequence. This human sequence (SEQ ID NO:3), as well as also the amino acid sequence (SEQ ID NO:4) derived therefrom, is also the subject of this Patent Specification. FIGS. 3b(1)-3b (2) also show the protein sequences of surface proteins for rat (rMeta-1, SEQ ID NO:2) human (hCD44, SEQ ID NO:6) and mouse (mCD44, SEQ ID NO:7) with extracellular regions in rat and human protein. rCD44 (SEQ ID NO:8) is also shown in this Figure.

Embodimental examples

Cells and antibodies

The following cloned Bsp cell lines were used for the investigation: BSp73 14ASML-1 and 10AS-7 and kept in culture as described by Matzku et al., (1983); furthermore, the mammary carcinoma cell lines described by Neri et al., (1982), monoclonal antibodies against BSp73 ASML membrane proteins were produced by immunisation of Balb c mice. After isolation of the spleen cells of an immunised mouse, these were fused with Ag8 myeloma cells for the immortalisation according to the method for the production of monoclonal antibodies of Kohler (1981). The then-obtained hybridoma cells were subjected to a screening process in order to find those which produce specific antibodies against BSp73ASML but not against BSp73AS and normal rat fibroblast cells. The precise procedure is described in the same way as by Matzku et al. (1989).

Monoclonal antibody (mAb)-producing hybridoma cells with the corresponding specificity were expanded in the tissue culture and the mAb given off into the medium highly enriched by ammonium sulphate precipitation and column chromatography (protein A-Sepharose and MonoQ) and used in this form for the investigations. One of them is mAb1.1ASML.

Immunofluorescence:

For the display of the variant CD44 molecule on different tumour cells, these were taken up in culture, then washed with phosphate-buffered common salt solution (PBS) and incubated with 1.1ASML for 30 minutes at 40° C. As secondary antibody for the detection of the binding, there was used a rhodamine-coupled rabbit anti-mouse IgG and shown in the fluorescence microscope.

Construction of the cDNA expression banks and immuno-screening.

PolyA+ RNA from BSp73ASML cells was "primed" with oligo (dT) and hexanucleotides of different composition and synthesised with reverse transcriptase from AMV of the first strand of the cDNA. The second strand of the cDNA was produced with E. coli DNA polymerase I, RNaseH and E. coli ligase and subsequently the doublestranded cDNA linearised on the ends with T4DNA. The vectors pEX1, 2 and 3 (Stanley and Luzio, 1984), which make possible the fusioning of the cDNA in 3 different reading rasters, were cleaved with SmaI restruction endonuclease and ligated with the cDNA (T4 DNA ligase). Competent E. coli DH5 (pCI857) bacteria, which produce a temperature-sensitive repressor, are transfected with the pEX-cDNA constructants and cultured on nylon filters. The gene for the temperature-sensitive repressor RCI857 lies on the plasmid pCI857, which is compatible with the pEX plasmids. At 28° C., the 1P_(R) promotor, which controls the synthesis of the fusion proteins, is inactivated. By temperature increase to 42° C., the CI repressor is inactivated and the synthesis of β-galactosidase/ASML fusion proteins massively set into action. The heat-induced bacteria colonies are subsequently denatured with chloroform vapour on the filters and these then incubated in PBS which contains 3% dry milk powder, lysozyme and DNase. The bacterial fusion proteins fixed on the nylon filter are now incubated with mAb1.1ASML and, after washing out of non-specifically-bound mAb, used for the detection of the binding as secondary antibody 125J-labelled rabbit anti-mouse IgG. After autoradiography, positive clones were isolated from the original bacteria filter and substantially analysed. One clone, which synthesised a fusion protein which reacted specifically with 1.1ASML, was pEX34. The pEX contained in the bacterial clone carries 167 nucleotide cDNA which, inter alia, codes for the epitope (or the antigen determinants), the specificity of which is carried by mAb1.1ASML.

The isolation of the total length cDNA mMeta-1 then took place according to standard methods.

Immunisation of the rats with mAb1.1ASML

BDX rats, which are syngenic to the BSp73 tumour cells, were injected subcutaneously or intraperitoneally with mAb1.1ASML (coupled to keyhole limpet haemocyananine), together with complete Freund's adjuvant. The first took place 10, 7 and 3 days before the injection of the BSpASML cells (into the fatty foot pad), the following then 3, 7, 11, 14 and 21 days thereafter. After 28 days, the rats were sacrificed, the various lymph nodes prepared and weighed and macroscopically visible lung metastases counted.

Connection between the expression of variant CD44 surface proteins and metastatic potential

In order to ascertain whether the expression of variant CD44 glucoproteins is merely a property of the investigated BSp73ASML cell line or whether the expression can be brought into connection with the metasiatic potential, another series of rat tumour cells, which are derived from the 13762NF mammary carcinoma (Neri et al., 1982), were investigated. Furthermore, cell lines which were derived from the primary tumours (MTPa, MTC, MTF7 and MTA (group 1)) were compared with cell lines which are derived from lymph nodes and lung metastases (MTLy, MTLn2, MTLn3 (group 2)). The pattern of the RNA derived from CD44 is given in FIGS. 4A-4G, whereby samples A, B and D correspond to the samples described on pages 5 and 6 of the Application, as well as to FIG. 1. Cells of group 1 all show a normal CD44 pattern with sample B. However, cells of group 2 show a pattern different therefrom. The RNA is larger than the RNA of group 1 and corresponds to the RNA of BSp73ASML. Smaller RNAs are lost in the case of the hybridisation with sample D. The other patterns show the similarity between the two rat tumour systems.

Also with the sample A, the RNA pattern of group 2 corresponds to that of BSp73ASML. Whereas sample A does not hybridise with RNA from BSp73AS, there is shown a small diffuse RNA band of about 2.5 kb in the case of cells of group 1. RNase and S1 protection analysis also show the structural similarity. From these results, an exchange in the cleavage pattern and the expression of variant CD44 RNAs appears to take place with the formation of metastases.

Transfer of the metastatic potential to non-metastasing BSp73AS cells by overexpression of pMeta-1.

The connection of the expression of variant CD44 species with the metastatic potential in two series of rat tumours indicates a causal role of the glycoproteins in the metastatic process. In order to investigate this, pMeta-1 was transferred into BSp73AS cells and investigated whether the behaviour of the cells is thereby changed. The complete coding region of the pMeta-1 (FIGS. 2a-2d, SEQ ID NO:1) was inserted below the SV40 promotor and this formation (diagram in FIG. 5) introduced into the BSp73AS cells, together with PSV2neo. Individual G418-resistant and pMeta-1expressing colonies were obtained. The RNA pattern of 2 of these colonies is shown in FIG. 5. The hybridisation of the variant CD44-specific sample A shows a dominant transcript of approximately 2.2 kb which corresponds to the size of the smallest frequent RNA which is transcribed in BSp73ASML cells (FIG. 5). However, the transfected cells contain about 10 times as much of this RNA as BSp73ASML.

Other size orders are observed in one of the transfected cells (BSp73AS-pSVMeta-1-14), which could be dependent upon the place of the plasmid integration. A pSV2neo simulation transfer clone (not shown) and the BSp73AS receiver cells contain no RNA which is complementary to sample A. In order to discover the endogenic normal CD44 transcriptions (without the extra domains of the pSVMeta-1) in the transfects, the filter was stripped and rehybridised with sample D. This part of the non-transferred 3' sequence is not contained in the expression clone (cf. FIG. 5). Sample D detects two main transcripts of 2.9 and 4.9 kb in the RNA of the two transfects (FIG. 5, right column), not only in the control BSp73AS but also in the non-illustrated BSp73ASpSVneo.

Approximate quantifications of the various agreeing hybridisations show that the transfects express approximately 5× as much of the variant CD44 RNAs, which are transcribed by the expression plasmid, as the endogenic gene transcripts.

The overexpressed cDNA is transferred into a protein. The two transfects, which are illustrated in FIG. 5, synthesise mAbl-immune-colourable proteins of the same apparent size, namely, a main product of 150 kDa and a weaker band at 100 kDa. Since the cDNA sequence codes a primary protein product of only 503 amino acids (corresponds to about 60,000 Dalton), all visible bands must represent modified forms. The 150 kDa band runs together with one of the modified forms of variant CD44 which is expressed in the metastasing cells BSp73ASML. BSp73AS or simulation-transferred BSp73ASpSVneo do not possess this protein. As in BSp73ASML cells, the epitope of the cells expressed by the transfects lies freely on the cell surface.

In order to demonstrate that the expression of variant CD44 suffices in order to impart a metastatic potential to BSp73AS cells, transfects were injected into syngenic BDX rats (spontaneous metastasis protocol). In earlier experiments, metastatic tumour cells BSp73ASML spread out quickly from the place of the injection and were completely distributed about 10 days after the injection (Matzku, 1984). All local tumours were, therefore, removed by amputations on the 10th day. All carriers of BSp73ASML cells and all animals which had been injected with an overexpressing transfect developed lung metastases (Table 1). The course of the metastasis formation was comparably quick within 5-8 weeks after the injection. Animals which had received Bsp73AS cells or simulation transfects were, after this time, completely healthy (apart from due to the amputation) and even after 5 months no metastases could be ascertained.

In spite of a surprising similarity in the strong metastasis formation, there are some interesting differences. In all animals, BSp73ASML cells reach the lymph nodes and lead to a massive enlargement of various nodes in the region of the inguinal groin and next to the aorta (Table 1). A transfect (BSp73AS-pSV meta-1-14) causes lymph node enlargement in 3 of 8 animals although all animals develop lung metastases (Table 1). No lymph node enlargement is ascertainable with the other transfect (BSp73AS-pSV Meta-1-15). The transfects appear, therefore, to be able to form colonies in the lungs without an obligatory growth phase of the lymph nodes.

The experiment according to Table 1 further points to another difference between BSp73AS transfects and BSp73ASML. The individual lung metastases are macroscopically visible, whereas those of BSp73ASML are small and numerous but, in a larger series with BSp73ASML (Reber et al., 1990), 11 of 20 animals develop 5-20 larger nodes per lung than the transfects.

In order to ascertain that the metastases formed were brought about by the injected transfects and in order to exclude the improbable possibility of a spontaneous mutation, which transfers a metastatic potential, the epitope-positive proteins in the total lung extracts and in the extracts of recultured metastasis-producing cells were determined. The 150 kDa glycoprotein is detectable in the whole lung extract as well as in the extracts of a specific lung node from an animal which has received BSpAS-pSV meta-1-15 transfects. In the case of in vitro growth, the G418-resistant strain expresses a protein of the same apparent molecular weight.

Diagnosis and therapy

1. Analysis of human tumour material by in situ hybridisation with the human pMeta-1 sequence present. These experiments are considered as preliminary experiments before an Ab is available which recognises the human ECR.

2. Production of antibodies against the human ECR. Cloning of the human pMeta-1 sequences in bacterial expression vectors so that fusions arise with β-galactosidase or tryptophane E-product. Immunisation of rabbits with these fusion proteins or with synthesised peptides from the ECR (coupled to carrier molecules). Isolation of the polyvalent or monospecific antibodies.

Possibilities of use:

Immunohistological investigations of clinical tumour material (diagnosis)

Detection of soluble ECR in the serum of patients with the help of ELISA tests (diagnosis)

Construction of toxin-coupled antibodies in order, with the help of the antibody, to bring the toxin into the tumour/metastasis region (therapy)

Construction of antibodies with two definite antigen binding positions. By means of this double specificity, the attempt is to be made to initiate cytotoxic reactions in the metastasis region (e.g. anti CD2 or CD3 coupling) (therapy).

3. Production of hMeta-1 protein by transfection of human or rat cells with an expression vector which carries the complete hMeta-1 cDNA sequence; or purification from LCLC97 cells.

Possibilities of use:

Injection of the protein or parts thereof in order to block the tissue binding positions of the tumour cells

After characterisation of the binding positions, a use for therapy would also be conceivable which could depend upon the injection of large amounts of binding protein which would then block the migrating tumour cells.

List of literature:

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Hart, I. R., Goode, N. T. and Wilson, R. E. (1989), Molecular aspects of the metastatic cascade, Biochim. Biophys. Acta 989: 65-84.

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Stanley K. K. and Luzio, J. P. (1984), Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins, EMBO J. 3: 1429-1434.

Wenzel, A. (1986), Charakterisierung von Differenzierungsantigenen auf dem Rattentumor Bsp 73 mit Hilfe monoklonaler Antikorper (Characterisation of differentiation antigens on the rat tumour Bsp 73 with the help of monoclonal antibodies), Diploma Dissertation, University of Karlsruhe.

Zhou, D. F. H., Ding, J. F., Picker, L. F., Bargatze, R. F., Butcher, E. C. and Goeddel, D. V. (1989), Molecular cloning and expression of Pgp-1 - The mouse homolog of the human H-CAM (Hermes) lymphocyte homing receptor, J. Immunol. 143: 3390-3395.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 8                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 3207 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: p-Meta-1                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 113..1624                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        CTCATTGCCCAGCAGCCCCCAGCCAGTGACAGGTTCCATTCACCCTCTTTGCCCCTTCCC60                 CCGCGACCCTTTTCCAGAGGCTACTAGATCCTTTGGTTTCATCCTGCACA TCATG115                    Met                                                                            GACAAGGTTTGGTGGCACACAGCTTGGGGACTACTTTGC CTCTTACAG163                           AspLysValTrpTrpHisThrAlaTrpGlyLeuLeuCysLeuLeuGln                               51015                                                                          TTGAGCCTGGCACAGCAGCAGATCGATTTGAATATAACC TGCCGTTAC211                           LeuSerLeuAlaGlnGlnGlnIleAspLeuAsnIleThrCysArgTyr                               202530                                                                         GCAGGTGTATTCCATGTGGAGAAAAATGGCCGCTACAGTATC TCCAGG259                           AlaGlyValPheHisValGluLysAsnGlyArgTyrSerIleSerArg                               354045                                                                         ACTGAAGCAGCTGACCTCTGCGAGGCTTTCAACACCACCTTGCCCACC 307                           ThrGluAlaAlaAspLeuCysGluAlaPheAsnThrThrLeuProThr                               50556065                                                                       ATGGCTCAGATGGAGTTAGCCCTGAGAAAGGGGTTTGAAACA TGCAGG355                           MetAlaGlnMetGluLeuAlaLeuArgLysGlyPheGluThrCysArg                               707580                                                                         TATGGGTTCATAGAAGGACACGTGGTAATCCCGAGGATC CACCCCAAC403                           TyrGlyPheIleGluGlyHisValValIleProArgIleHisProAsn                               859095                                                                         GCTATCTGTGCAGCCAACAACACAGGAGTGTATATCCTC CTCGCATCC451                           AlaIleCysAlaAlaAsnAsnThrGlyValTyrIleLeuLeuAlaSer                               100105110                                                                      AACACCTCCCACTATGACACATATTGCTTCAATGCCTCAGCT CCTCTT499                           AsnThrSerHisTyrAspThrTyrCysPheAsnAlaSerAlaProLeu                               115120125                                                                      GAAGAAGACTGTACATCAGTCACAGACCTACCCAATTCCTTCGATGGA 547                           GluGluAspCysThrSerValThrAspLeuProAsnSerPheAspGly                               130135140145                                                                   CCAGTTACCATAACTATTGTCAACCGTGATGGCACCCGCTAC AGCAAG595                           ProValThrIleThrIleValAsnArgAspGlyThrArgTyrSerLys                               150155160                                                                      AAGGGCGAGTATAGAACACACCAAGAAGACATCGATGCC TCAAACATT643                           LysGlyGluTyrArgThrHisGlnGluAspIleAspAlaSerAsnIle                               165170175                                                                      ATAGATGAGGATGTCAGCAGTGGATCCACCATTGAGAAG AGCACCCCA691                           IleAspGluAspValSerSerGlySerThrIleGluLysSerThrPro                               180185190                                                                      GAAGGCTACATTTTGCACACCGACCTTCCCACTTCACAGCCT ACTGGA739                           GluGlyTyrIleLeuHisThrAspLeuProThrSerGlnProThrGly                               195200205                                                                      GACCGGGATGACGCCTTCTTTATTGGGAGCACCCTGGCCACCATTGCA 787                           AspArgAspAspAlaPhePheIleGlySerThrLeuAlaThrIleAla                               210215220225                                                                   ACTACTCCATGGGTTTCTGCCCACACAAAACAGAACCAGGAA CGGACC835                           ThrThrProTrpValSerAlaHisThrLysGlnAsnGlnGluArgThr                               230235240                                                                      CAGTGGAACCCGATCCATTCAAACCCAGAAGTACTACTT CAGACAACC883                           GlnTrpAsnProIleHisSerAsnProGluValLeuLeuGlnThrThr                               245250255                                                                      ACCAGGATGACTGATATAGACAGAAACAGCACCAGTGCT CATGGAGAA931                           ThrArgMetThrAspIleAspArgAsnSerThrSerAlaHisGlyGlu                               260265270                                                                      AACTGGACCCAGGAACCACAGCCTCCTTTCAATAACCATGAG TATCAG979                           AsnTrpThrGlnGluProGlnProProPheAsnAsnHisGluTyrGln                               275280285                                                                      GATGAAGAGGAGACCCCACATGCTACAAGCACAACCTGGGCAGATCCT 1027                          AspGluGluGluThrProHisAlaThrSerThrThrTrpAlaAspPro                               290295300305                                                                   AATAGCACAACAGAAGAAGCAGCTACCCAGAAGGAGAAGTGG TTTGAG1075                          AsnSerThrThrGluGluAlaAlaThrGlnLysGluLysTrpPheGlu                               310315320                                                                      AATGAATGGCAGGGGAAGAACCCACCCACCCCAAGTGAA GACTCCCAT1123                          AsnGluTrpGlnGlyLysAsnProProThrProSerGluAspSerHis                               325330335                                                                      GTGACAGAAGGGACAACTGCCTCAGCCCACAACAACCAT CCAAGTCAA1171                          ValThrGluGlyThrThrAlaSerAlaHisAsnAsnHisProSerGln                               340345350                                                                      AGAATGACAACACAGAGTCAAGAGGATGTTTCATGGACCGAT TTCTTC1219                          ArgMetThrThrGlnSerGlnGluAspValSerTrpThrAspPhePhe                               355360365                                                                      GACCCAATCTCACATCCAATGGGACAAGGTCATCAAACAGAAAGCAAG 1267                          AspProIleSerHisProMetGlyGlnGlyHisGlnThrGluSerLys                               370375380385                                                                   GGACACTCAAGTGGGAATCAAGACAGTGGAGTGACCACAACT TCTGGT1315                          GlyHisSerSerGlyAsnGlnAspSerGlyValThrThrThrSerGly                               390395400                                                                      CCTGCGAGGAGACCTCAGATTCCAGAGTGGCTTATCATC TTGGCATCC1363                          ProAlaArgArgProGlnIleProGluTrpLeuIleIleLeuAlaSer                               405410415                                                                      CTCCTGGCGCTGGCTCTGATTCTTGCCGTCTGCATTGCT GTCAACAGT1411                          LeuLeuAlaLeuAlaLeuIleLeuAlaValCysIleAlaValAsnSer                               420425430                                                                      AGGAGAAGGTGTGGGCAGAAGAAGAAGCTGGTGATCAACAGT GGCAAT1459                          ArgArgArgCysGlyGlnLysLysLysLeuValIleAsnSerGlyAsn                               435440445                                                                      GGAACAGTGGAAGACAGGAAACCAAGTGAACTCAACGGGGAGGCCAGC 1507                          GlyThrValGluAspArgLysProSerGluLeuAsnGlyGluAlaSer                               450455460465                                                                   AAGTCTCAGGAAATGGTGCATTTGGTGAACAAGGAACCAACA GAGACT1555                          LysSerGlnGluMetValHisLeuValAsnLysGluProThrGluThr                               470475480                                                                      CCGGACCAGTTTATGACAGCTGATGAGACCCGGAATCTG CAGAGTGTG1603                          ProAspGlnPheMetThrAlaAspGluThrArgAsnLeuGlnSerVal                               485490495                                                                      GATATGAAGATTGGGGTGTAGTGCCTATGCCACTAACTTGAAA AGACA1651                          AspMetLysIleGlyVal                                                             500                                                                            CAACAATTGGAGACATGTCATTACTGGGAGCTGGGACCCTTAACAGATGCAATGTGCTAC1711               TGATTATTTTTTATTGGGATTATTTTGGGCATAAAATTTCCCTTTTTTTGTTTT TTAAAA1771              GTTTGTTTTCCAATTTATGAAAATAGCATTGCTTTCTGAAATGAGGGTCTCTTCCAGTTC1831               CTCCTTAGAGGCCTTGCATTACCAGGGTATGCTACCATAGGCTTCTACCAAATGAATACT1891               CTTGGTCCCGATTGAACCCAAAGTCCCAG GTAACATCCACCAGCTAAGGATTTCCCCAGA1951              ACTTAGAGAGATTGGTCTCTGGGAGGAAATTTGAATGGGTCCATATTGCCTCCCAGCAGT2011               CCAATCTGTAGGCATTGCTTTGCAGTGGATGGGAGATCAGGTGTACTGGTTACACACTCT2071               CTT TATAGACTCCCTTCTGCTGGAAAATTTCCACATGCTTCTGAGAGATTCCCCAAAGGT2131              GACGCTATTTATCTTTAGTAAGCTATTTATCTTTGTTTTTGAAATATCAAACCCTGGAGG2191               TCCTTTTTTCAGTATGACTTTTTTTATTTTGTTTTTTTTTATTTTGT TTTTTAGGTTACT2251              TTGTCAGAAGCATAACAGGGTATAAGTTGATTCATAATAAATACCTGTCCATCTTCCATC2311               TTGACCTGTTGTGCTGTGATCCTTCAGTTTCTAAATCAGCAAGGTCTGAGTCTTTGTAGC2371               ACATCAATGTGACCTTAGTAT GGTCCTCTGAAACTCATGTTAGAGCATCCGTGCCCTGCT2431              TGGGTTTACCCAGCTGAATCTCAGAAGATCAAGGACAGGAGCACTGTTTTCATTCTAGGA2491               CTATCAAAGGGGTTTCTCTCCTGTTCAAGAATCTGAATTGGGAGTAGGAGAGCTTCTGTC255 1              CCTTTTATGTTTCGATAACCACCCATTTCTCTTTCTTAAAGGGCACATTAAGTTTTTATA2611               TCTTACAACATTCGCGGTCCTGTTTCATAGACACTGATCTTATTGGCACTTTCACAAAAC2671               AGTGTGGAGGGGACTTCTGACACCTTATAGTAAAAGGAGA AGCCAACAGAAATGAAAGTG2731              TGGACAGAGAGCAGTAGATTGGCATGAGGAGGCATGATGTACAACCCCCAGACCACTCTT2791               TCCATCACCACATTTGTTGATGCTTTCGCAAGCCAGTTGGTACTTAGAATCAGTTCCCCA2851               GGGAATCCTTCAAA AAGCCATAAGAATGCCCACCCCTGGAATCTTACCACCACCAGATGA2911              GCAGGTTTATGGTTTAGCAAAAGGAGAATGCTGTCACCCTCTGACCTCATAGTTTTCACA2971               TACTGGGCAAGTGTTCATCTGCCAGGATGCCCCATTGCTCCTAGGTCTTCCCAGGTAC CT3031              TGTAGAAGAACTTAAATCTATAAAATAAGGCTTTCTCTAAAATGGAACTTCCTTTCTAAG3091               GCTCCCATTTTTACTGTTGACTAAATTTATATGTTTAATAGTTTTTTTTCAAATAAAAAC3151               AAACACAAAAAGGAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA3207                  (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 503 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetAspLysValTrpTrpHisThrAlaTrpGly LeuLeuCysLeuLeu                              151015                                                                         GlnLeuSerLeuAlaGlnGlnGlnIleAspLeuAsnIleThrCysArg                               2025 30                                                                        TyrAlaGlyValPheHisValGluLysAsnGlyArgTyrSerIleSer                               354045                                                                         ArgThrGluAlaAlaAspLeuCysGluAlaPheAsnThrThrLeuPro                                505560                                                                        ThrMetAlaGlnMetGluLeuAlaLeuArgLysGlyPheGluThrCys                               65707580                                                                       ArgTyrGlyPheIl eGluGlyHisValValIleProArgIleHisPro                              859095                                                                         AsnAlaIleCysAlaAlaAsnAsnThrGlyValTyrIleLeuLeuAla                               100 105110                                                                     SerAsnThrSerHisTyrAspThrTyrCysPheAsnAlaSerAlaPro                               115120125                                                                      LeuGluGluAspCysThrSerValThrAspLeu ProAsnSerPheAsp                              130135140                                                                      GlyProValThrIleThrIleValAsnArgAspGlyThrArgTyrSer                               145150155 160                                                                  LysLysGlyGluTyrArgThrHisGlnGluAspIleAspAlaSerAsn                               165170175                                                                      IleIleAspGluAspValSerSerGlySerThrIleGluLysSerThr                               180185190                                                                      ProGluGlyTyrIleLeuHisThrAspLeuProThrSerGlnProThr                               195200205                                                                      GlyAspArgAspAs pAlaPhePheIleGlySerThrLeuAlaThrIle                              210215220                                                                      AlaThrThrProTrpValSerAlaHisThrLysGlnAsnGlnGluArg                               225230 235240                                                                  ThrGlnTrpAsnProIleHisSerAsnProGluValLeuLeuGlnThr                               245250255                                                                      ThrThrArgMetThrAspIleAspArgAsn SerThrSerAlaHisGly                              260265270                                                                      GluAsnTrpThrGlnGluProGlnProProPheAsnAsnHisGluTyr                               275280 285                                                                     GlnAspGluGluGluThrProHisAlaThrSerThrThrTrpAlaAsp                               290295300                                                                      ProAsnSerThrThrGluGluAlaAlaThrGlnLysGluLysTrpPhe                               305 310315320                                                                  GluAsnGluTrpGlnGlyLysAsnProProThrProSerGluAspSer                               325330335                                                                      HisValThrGl uGlyThrThrAlaSerAlaHisAsnAsnHisProSer                              340345350                                                                      GlnArgMetThrThrGlnSerGlnGluAspValSerTrpThrAspPhe                               355 360365                                                                     PheAspProIleSerHisProMetGlyGlnGlyHisGlnThrGluSer                               370375380                                                                      LysGlyHisSerSerGlyAsnGlnAspSerGlyValThr ThrThrSer                              385390395400                                                                   GlyProAlaArgArgProGlnIleProGluTrpLeuIleIleLeuAla                               405410 415                                                                     SerLeuLeuAlaLeuAlaLeuIleLeuAlaValCysIleAlaValAsn                               420425430                                                                      SerArgArgArgCysGlyGlnLysLysLysLeuValIleAsnSerGly                               435440445                                                                      AsnGlyThrValGluAspArgLysProSerGluLeuAsnGlyGluAla                               450455460                                                                      SerLysSerGlnGluMetVa lHisLeuValAsnLysGluProThrGlu                              465470475480                                                                   ThrProAspGlnPheMetThrAlaAspGluThrArgAsnLeuGlnSer                               485 490495                                                                     ValAspMetLysIleGlyVal                                                          500                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1062 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: human cDNA                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1062                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        GTACACCCCATCCCAGACGAAGACAGTCCCTGGATCACCGACAGCACA48                             ValHisProIleProAsp GluAspSerProTrpIleThrAspSerThr                              151015                                                                         GACAGAATCCCTGCTACCACAGGCTGGGAGCCAAATGAAGAAAATGAA96                             AspArgIleProAla ThrThrGlyTrpGluProAsnGluGluAsnGlu                              202530                                                                         GATGAAAGAGACAGACACCTCAGTTTTTCTGGATCAGGCATTGATGAT144                            AspGluArgAspArg HisLeuSerPheSerGlySerGlyIleAspAsp                              354045                                                                         GATGAAGATTTTATCTCCAGCACCATTTCAACCACACCACGGGCCTTT192                            AspGluAspPheIleSer SerThrIleSerThrThrProArgAlaPhe                              505560                                                                         GACCACACAAAACAGAACCAGGACTGGACCCAGTGGAACCCAAGCCAT240                            AspHisThrLysGlnAsnGlnAsp TrpThrGlnTrpAsnProSerHis                              65707580                                                                       TCAAATCCGGAAGTGCTACTTCAGACAACCACAAGGATGACTGATGTA288                            SerAsnProGluValLeu LeuGlnThrThrThrArgMetThrAspVal                              859095                                                                         GACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCA336                            AspArgAsnGlyThr ThrAlaTyrGluGlyAsnTrpAsnProGluAla                              100105110                                                                      CACCCTCCCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCA384                            HisProProLeuIle HisHisGluHisHisGluGluGluGluThrPro                              115120125                                                                      CATTCTACAAGCACAATCCAGGCAACTCCTAGTAGTACAACGGAAGAA432                            HisSerThrSerThrIle GlnAlaThrProSerSerThrThrGluGlu                              130135140                                                                      ACAGCTACCCAGAAGGAACAGTGGTTTGGCAACAGATGGCATGAGGGA480                            ThrAlaThrGlnLysGluGlnTrp PheGlyAsnArgTrpHisGluGly                              145150155160                                                                   TATCGCCAAACACCCAGAGAAGACTCCCATTCGACAACAGGGACAGCT528                            TyrArgGlnThrProArg GluAspSerHisSerThrThrGlyThrAla                              165170175                                                                      GCAGCCTCAGCTCATACCAGCCATCCAATGCAAGGAAGGACAACACCA576                            AlaAlaSerAlaHis ThrSerHisProMetGlnGlyArgThrThrPro                              180185190                                                                      AGCCCAGAGGACAGTTCCTGGACTGATTTCTTCAACCCAATCTCACAC624                            SerProGluAspSer SerTrpThrAspPhePheAsnProIleSerHis                              195200205                                                                      CCCATGGGACGAGGTCATCAAGCAGGAAGAAGGATGGATATGGACTCC672                            ProMetGlyArgGlyHis GlnAlaGlyArgArgMetAspMetAspSer                              210215220                                                                      AGTCATAGTACAACGCTTCAGCCTACTGCAAATCCAAACACAGGTTTG720                            SerHisSerThrThrLeuGlnPro ThrAlaAsnProAsnThrGlyLeu                              225230235240                                                                   GTGGAAGATTTGGACAGGACAGGACCTCTTTCAATGACAACGCAGCAG768                            ValGluAspLeuAspArg ThrGlyProLeuSerMetThrThrGlnGln                              245250255                                                                      AGTAATTCTCAGAGCTTCTCTACATCACATGAAGGCTTGGAAGAAGAT816                            SerAsnSerGlnSer PheSerThrSerHisGluGlyLeuGluGluAsp                              260265270                                                                      AAAGACCATCCAACAACTTCTACTCTGACATCAAGCAATAGGAATGAT864                            LysAspHisProThr ThrSerThrLeuThrSerSerAsnArgAsnAsp                              275280285                                                                      GTCACAGGTGGAAGAAGAGACCCAAATCATTCTGAAGGCTCAACTACT912                            ValThrGlyGlyArgArg AspProAsnHisSerGluGlySerThrThr                              290295300                                                                      TTACTGGAAGGTTATACCTCTCATTACCCACACACGAAGGAAAGCAGG960                            LeuLeuGluGlyTyrThrSerHis TyrProHisThrLysGluSerArg                              305310315320                                                                   ACCTTCATCCCAGTGACCTCAGCTAAGACTGGGTCCTTTGGAGTTACT1008                           ThrPheIleProValThr SerAlaLysThrGlySerPheGlyValThr                              325330335                                                                      GCAGTTACTGTTGGAGATTCCAACTCTAATGTCAATCGTTCCTTATCA1056                           AlaValThrValGly AspSerAsnSerAsnValAsnArgSerLeuSer                              340345350                                                                      GGAGAC1062                                                                     GlyAsp                                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 354 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        ValHisProIleProAspGluAspSerProTrpIleThrAspSerThr                               15 1015                                                                        AspArgIleProAlaThrThrGlyTrpGluProAsnGluGluAsnGlu                               202530                                                                         AspGluArgAspArgHisLeuSerPheSer GlySerGlyIleAspAsp                              354045                                                                         AspGluAspPheIleSerSerThrIleSerThrThrProArgAlaPhe                               505560                                                                         A spHisThrLysGlnAsnGlnAspTrpThrGlnTrpAsnProSerHis                              65707580                                                                       SerAsnProGluValLeuLeuGlnThrThrThrArgMetThrAspVal                                859095                                                                        AspArgAsnGlyThrThrAlaTyrGluGlyAsnTrpAsnProGluAla                               100105110                                                                      HisProProLe uIleHisHisGluHisHisGluGluGluGluThrPro                              115120125                                                                      HisSerThrSerThrIleGlnAlaThrProSerSerThrThrGluGlu                               130135 140                                                                     ThrAlaThrGlnLysGluGlnTrpPheGlyAsnArgTrpHisGluGly                               145150155160                                                                   TyrArgGlnThrProArgGluAspSerHisSer ThrThrGlyThrAla                              165170175                                                                      AlaAlaSerAlaHisThrSerHisProMetGlnGlyArgThrThrPro                               180185 190                                                                     SerProGluAspSerSerTrpThrAspPhePheAsnProIleSerHis                               195200205                                                                      ProMetGlyArgGlyHisGlnAlaGlyArgArgMetAspMetAspSer                                210215220                                                                     SerHisSerThrThrLeuGlnProThrAlaAsnProAsnThrGlyLeu                               225230235240                                                                   ValGluAspLeuAs pArgThrGlyProLeuSerMetThrThrGlnGln                              245250255                                                                      SerAsnSerGlnSerPheSerThrSerHisGluGlyLeuGluGluAsp                               260 265270                                                                     LysAspHisProThrThrSerThrLeuThrSerSerAsnArgAsnAsp                               275280285                                                                      ValThrGlyGlyArgArgAspProAsnHisSer GluGlySerThrThr                              290295300                                                                      LeuLeuGluGlyTyrThrSerHisTyrProHisThrLysGluSerArg                               305310315 320                                                                  ThrPheIleProValThrSerAlaLysThrGlySerPheGlyValThr                               325330335                                                                      AlaValThrValGlyAspSerAsnSerAsnValAsnArgSerLeuSer                               340345350                                                                      GlyAsp                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 355 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: rat protein                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        SerGlnProThrGlyAspArgAspAspAlaPhePheIleGlySerThr                               151015                                                                         LeuAlaThrSerThrGluSerAsnThrAsnProThrGly TrpLysPro                              202530                                                                         AsnGluGluAsnGluAspGluThrAspLysTyrProAsnPheSerGly                               3540 45                                                                        SerGlyIleAspAspAspGluAspPheIleSerSerThrIleAlaThr                               505560                                                                         ThrProTrpValSerAlaHisThrLysGlnAsnGlnGluArgThrG ln                              65707580                                                                       TrpAsnProIleHisSerAsnProGluValLeuLeuGlnThrThrThr                               8590 95                                                                        ArgMetThrAspIleAspArgAsnSerThrSerAlaHisGlyGluAsn                               100105110                                                                      TrpThrGlnGluProGlnProProPheAsnAsnHi sGluTyrGlnAsp                              115120125                                                                      GluGluGluThrProHisAlaThrSerThrThrTrpAlaAspProAsn                               130135 140                                                                     SerThrThrGluGluAlaAlaThrGlnLysGluLysTrpPheGluAsn                               145150155160                                                                   GluTrpGlnGlyLysAsnProProThrProSerG luAspSerHisVal                              165170175                                                                      ThrGluGlyThrThrAlaSerAlaHisAsnAsnHisProSerGlnArg                               180185 190                                                                     MetThrThrGlnSerGlnGluAspValSerTrpThrAspPhePheAsp                               195200205                                                                      ProIleSerHisProMetGlyGlnGlyHis GlnThrGluSerLysAsp                              210215220                                                                      ThrGlySerSerHisSerThrThrLeuGlnProThrAlaAlaProAsn                               22523023 5240                                                                  ThrHisLeuValGluAspLeuAsnArgThrGlyProLeuSerValThr                               245250255                                                                      ThrProGlnSerHisSerGlnAsn PheSerThrLeuProGlyGluLeu                              260265270                                                                      GluGluGlyGluAspHisProThrThrSerValLeuProSerSerThr                               275 280285                                                                     LysSerGlyArgArgArgGlyGlySerLeuProArgAspThrThrThr                               290295300                                                                      SerLeuGluGlyTyrThrProGlnTyrPr oAspThrMetGluAsnGly                              305310315320                                                                   ThrLeuPheProValThrProAlaLysThrGluValPheGlyGluThr                               325 330335                                                                     GluGlyThrValAlaThrAspSerAsnPheAsnValAspGlySerLeu                               340345350                                                                      ProGlyAsp                                                                       355                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 361 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: hCD44                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetAspLysPheTrpTrpHisAlaAlaTrpGlyLeuC ysLeuValPro                              151015                                                                         LeuSerLeuAlaGlnIleAspLeuAsnIleThrCysArgPheAlaGly                               2025 30                                                                        ValPheHisValGluLysAsnGlyArgTyrSerIleSerArgThrGlu                               354045                                                                         AlaAlaAspLeuCysLysAlaPheAsnSerThrLeu ProThrMetAla                              505560                                                                         GlnMetGluLysAlaLeuSerIleGlyPheGluThrCysArgTyrGly                               657075 80                                                                      PheIleGluGlyHisValValIleProArgIleHisProAsnSerIle                               859095                                                                         CysAlaAlaAsnAsnThrGlyValTyrIleL euThrTyrAsnThrSer                              100105110                                                                      GlnTyrAspThrTyrCysPheAsnAlaSerAlaProProGluGluAsp                               115120 125                                                                     CysThrSerValThrAspLeuProAsnAlaPheAspGlyProIleThr                               130135140                                                                      IleThrIleValAsnArgAspGlyThrArgTyrVal GlnLysGlyGlu                              145150155160                                                                   TyrArgThrAsnProGluAspIleTyrProSerAsnProThrAspAsp                               165 170175                                                                     AspValSerSerGlySerSerSerGluArgSerSerThrSerGlyGly                               180185190                                                                      TyrIlePheTyrThrPheSerThr ValHisProIleProAspGluAsp                              195200205                                                                      SerProTrpIleThrAspSerThrAspArgIleProAlaThrArgAsp                               210215 220                                                                     GlnAspThrPheHisProSerGlyGlySerHisThrThrHisGluSer                               225230235240                                                                   GluSerAspGlyHisSerHisGl ySerGlnGluGlyGlyAlaAsnThr                              245250255                                                                      ThrSerGlyProIleArgThrProGlnIleProGluTrpLeuIleIle                               260 265270                                                                     LeuAlaSerLeuLeuAlaLeuAlaLeuIleLeuAlaValCysIleAla                               275280285                                                                      ValAsnSerArgArgArgC ysGlyGlnLysLysLysLeuValIleAsn                              290295300                                                                      SerGlyAsnGlyAlaValGluAspArgLysProSerGlyLeuAsnGly                               305310 315320                                                                  GluAlaSerLysSerGlnGluMetValHisLeuValAsnLysGluSer                               325330335                                                                      SerGluThrPro AspGlnPheMetThrAlaAspGluThrArgAsnLeu                              340345350                                                                      GlnAsnValAspMetLysIleGlyVal                                                    355360                                                                         (2 ) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 363 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: mCD44                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        MetAspLysPheTrpTrpHisThrAlaTrpGlyLeuCysLeuLeuGln                                151015                                                                        LeuSerLeuAlaHisGlnGlnIleAspLeuAsnValThrCysArgTyr                               202530                                                                         AlaGlyValPheCysValGluLysAsnGlyArgTyrSerIleSerArg                               354045                                                                         ThrGluAlaAlaAspLeuCysGlnAlaPheAsnSerThrLeuProThr                               505560                                                                         MetAspGlnMetLysLeuAlaLeuSerLysGlyPheGluThrCysArg                               65707580                                                                        TyrGlyPheIleGluGlyAsnValValIleProArgIleHisProAsn                              859095                                                                         AlaIleCysAlaAlaAsnHisThrGlyValTyrIleLeuValT hrSer                              100105110                                                                      AsnThrSerHisTyrAspThrTyrCysPheAsnAlaSerAlaProPro                               115120125                                                                      GluGluAspCysThrSerValThrAspLeuProAsnSerPheAspGly                               130135140                                                                      ProValThrIleThrIleValAsnArgAspGlyThrArgTyrSerLys                               145150155160                                                                   LysGlyGluTyrArgThrHisGlnGluAspIleAspAlaSerAsnIle                               165170 175                                                                     IleAspAspAspValSerSerGlySerThrIleGluLysSerThrPro                               180185190                                                                      GluGlyTyrIleLeuHisThrTyrLeuProThrGlu GlnProThrGly                              195200205                                                                      AspGlnAspAspSerPhePheIleArgSerThrLeuAlaThrArgAsp                               2102152 20                                                                     ArgAspSerSerLysAspSerArgGlySerSerArgThrValThrHis                               225230235240                                                                   GlySerGluLeuAlaGlyHisSerSerAlaAsnGl nAspSerGlyVal                              245250255                                                                      ThrThrThrSerGlyProMetArgArgProGlnIleProGluTrpLeu                               260265 270                                                                     IleIleLeuAlaSerLeuLeuAlaLeuAlaLeuIleLeuAlaValCys                               275280285                                                                      IleAlaValAsnSerArgArgArgCysGlyG lnLysLysLysLeuVal                              290295300                                                                      IleAsnGlyGlyAsnGlyThrValGluAspArgLysProSerGluLeu                               305310315 320                                                                  AsnGlyGluAlaSerLysSerGlnGluMetValHisLeuValAsnLys                               325330335                                                                      GluProSerGluThrProAspGln CysMetThrAlaAspGluThrArg                              340345350                                                                      AsnLeuGlnSerValAspMetLysIleGlyVal                                              355360                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: rCD44                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        SerAspGlyAspSerSerMetAspProArgGlyGlyPheAspThrVal                               1 51015                                                                        ThrHisGlySerGluLeuAla                                                          20                                                                        

                  TABLE 1                                                          ______________________________________                                         Metastatic spreading out of BSp73AS cells which express                        variant CD44 cDNA pMeta-1***                                                           local  distribution in the case                                                appear-                                                                               of metastatic autopsy                                           tumour clone                                                                             ance     *LN ing   *LN par lung                                      ______________________________________                                         BSp73ASML 0/8      8/8       8/8     8/8                                                          0 1.5-2.5**                                                                              0 2.5-5.0                                                                              miliary                                   BSp73AS-  0/8      3/8       3/8     8/8                                       pSVMeta-1-14       0 0.3-1.2 0 1.0-4.5                                                                              multiple                                                                       0 0.3-5.0                                 BSp73AS-  0/8      0/8       0/8     8/8                                       pSVMeta-1-15                          5-20                                                                           0 0.3-10.0                               BSp73AS   1/8      0/8       0/8     0/8                                       BSp73AS-  0/8      0/8       0/8     0/8                                       pSVneo                                                                         ______________________________________                                          **average diameter in mm                                                       ***the Table gives the stage 60 days after injection of the given cells        *LN = lymph nodes                                                         

We claim:
 1. An isolated DNA fragment encoding all or part of a surface protein of metastasizing tumor cells, wherein said DNA fragment comprises a nucleotide sequence selected from the group consisting of:

    ______________________________________                                         (a) ATT GCA ACT ACT CCA TGG GTT TCT GCC CAC                                    ACA AAA CAG AAC CAG GAA CGG ACC CAG TGG                                        AAC CCG ATC CAT TCA AAC CCA GAA GTA CTA CTT                                    CAG ACA ACC ACC AGG ATG ACT GAT ATA GAC AGA                                    AAC AGC ACC AGT GCT CAT GGA GAA AAC TGG ACC                                    CAG GAA CCA CAG CCT CCT TTC AAT AAC CAT GAG                                    TAT CAG GAT GAA GAG GAG ACC CCA CAT GCT ACA                                    AGC ACA ACC TGG GCA GAT CCT AAT AGC ACA ACA                                    GAA GAA GCA GCT ACC CAG AAG GAG AAG TGG                                        TTT GAG AAT GAA TGG CAG GGG AAG AAC CCA                                        CCC ACC CCA AGT GAA GAC TCC CAT GTG ACA                                        GAA GGG ACA ACT GCC TCA GCC CAC AAC AAC                                        CAT CCA AGT CAA AGA ATG ACA ACA CAG AGT                                        CAA GAG GAT GTT TCA TGG ACC GAT TTC TTC                                        GAC CCA ATC TCA CAT CCA ATG GGA CAA (bases                                     782-1246 of SEQ ID NO: 1) and                                                  (b) AAC CCA AGC CAT TCA AAT CCG GAA GTG CTA                                    CTT CAG ACA ACC ACA AGG ATG ACT GAT GTA GAC                                    AGA AAT GGC ACC ACT GCT TAT GAA GGA AAC TGG                                    AAC CCA GAA GCA CAC CCT CCC CTC ATT CAC CAT                                    GAG CAT CAT GAG GAA GAA GAG ACC CCA CAT TCT                                    ACA AGC ACA ATC CAG GCA ACT CCT AGT AGT ACA                                    ACG GAA GAA ACA GCT ACC CAG AAG GAA CAG                                        TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC                                    CAA ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA                                    GGG ACA GCT GCA GCC TCA GCT CAT ACC AGC CAT                                    CCA ATG CA (bases 229-560 of SEQ ID NO: 3).                                    ______________________________________                                    


2. A recombinant DNA molecule, comprising a vector and a DNA fragment according to claim
 1. 3. A recombinant DNA molecule according to claim 2, wherein said vector is an expression vector.
 4. A transformed host cell comprising a DNA fragment according to claim
 1. 5. A transformed host cell comprising a recombinant DNA molecule according to claim
 2. 